Visualization of gene expression in Pristionchus pacificus with smFISH and in situ HCR

Single molecule fluorescence in situ hybridization (smFISH) and in situ hybridization chain reaction (HCR) have become powerful tools to visualize gene expression in many different animal species. We show here that smFISH and in situ HCR can be put to effective use in the satellite nematode model organism Pristionchus pacificus . Examining the expression of a homeobox gene ( Ppa-unc-30) , we found that HCR is more sensitive than smFISH. We confirmed the robustness of HCR by visualization of the expression of several genes involved in neurotransmitter synthesis or transport ( Ppa-unc-25 /GAD, Ppa-unc-17/VAChT, Ppa-eat-4 /VGLUT). Combined with its relative cost-effectiveness compared to smFISH analysis, in situ HCR constitutes a useful addition to the toolbox for P. pacificus research .


Description
The nematode Pristionchus pacificus is a powerful satellite model organism for comparative evolutionary studies (Sommer and McGaughran 2013).One key component to the success of comparative analysis is the ability to reliably assess gene expression patterns with spatial and temporal specificity in whole animals.While fluorescent reporter transgenes can be generated with ease in C. elegans, they are more difficult to generate in P. pacificus, even though substantial strides have been made in improving the efficiency of P. pacificus transgenesis (Schlager et al. 2009 Single molecule fluorescence in situ hybridization (smFISH) represents an alternative means to visualize gene expression (Ji and van Oudenaarden 2012), but the high costs of probe generation restrict the attractiveness of this technique.The more recent introduction of in situ hybridization chain reaction (HCR) represent a more cost-effective approach for the in situ analysis of gene expression (Choi et al. 2016).The greater cost-effectiveness stems from the ability to use unconjugated oligonucleotide sequences as probes.Each probe set consists of 25bp probes generated from complementary sequences within a gene's locus and a 18bp initiator sequence unique to an amplifier (B1, B2, B3, etc.).The fluorophores used to visualize probe-target hybridization are conjugated to a hairpin sequence that is not gene-specific and thus may be used for multiple experiments.HCR has been applied in many different organisms, including C. elegans (Choi et al. 2016) and we set out here to test the usage of HCR, as well as of smFISH, in P. pacificus.
We generated smFISH probes for two P. pacificus genes, Ppa-unc-25 (GAD), encoding glutamic acid decarboxylase, the rate limiting enzyme for synthesis of the neurotransmitter GABA (Eastman et al. 1999) and Ppa-unc-30, a Pitx-type homeobox transcription factor whose C. elegans ortholog controls Ppa-unc-25 expression in GABAergic ventral nerve cord neurons (Jin et al. 1994).We were able to readily detect Ppa-unc-25 transcripts in a pattern very similar to what is observed in C. elegans, namely in the ventral nerve cord and in several head neurons, including what appear to be the RME, RIS, RIB and AVL neurons (Figure 1A).However, we failed to detect any signals with smFISH probes for the Ppa-unc-30 locus.Moreover, Ppaunc-25 smFISH probing was unstable, requiring worms to be immediately imaged following the protocol to avoid risk of losing signal altogether.
We generated HCR probes for the same two genes (Ppa-unc-25, Ppa-unc-30) and followed a protocol that has been put to use in C. elegans (see Methods section) (Choi et al. 2016).We observed more robust signals with the Ppa-unc-25 HCR probes, and we now also observed signals with the Ppa-unc-30 HCR probes in the ventral nerve cord, as well as several head neurons (Figure 1B).
We further probed the generality of HCR usage by generating probes to two other genes, the single Ppa-eat-4/VGLUT locus to reveal glutamatergic neurons and the single Ppa-unc-17/VAChT locus to reveal cholinergic neurons.We generated these two probes with different amplifier sequences to allow us to co-stain the expression of each gene relative to the expression of Ppaunc-25/GAD.As expected from studies in C. elegans (Serrano-Saiz et al. but future studies will be required to pinpoint the exact identity of the Ppa-eat-4/VGLUT and Ppa-unc-17/VAChT expressing neurons.Since mRNA signals are unevenly distributed throughout the soma of a given cell, it is difficult to separate signals from neighboring cells within densely packed ganglia.We envision that the problem of cell identification will be solved by multicolor co-staining with co-expressed genes.
We note that smFISH and HCR appear to offer several advantages over antibody staining.Due to antibody penetration issues, immunostaining protocols usually only stain a fraction of the animals in the sample, often with a strong bias for larval stage animals.On the other hand, we note that smFISH and HCR protocols produce signals in nearly 100% of larval and adult stage animals.Additionally, many antibody staining protocols require harsh steps to fix and permeabilize the C. elegans cuticle (Duerr 2006 In conclusion, the cost-effectiveness, robustness, and sensitivity of the HCR protocol will empower P. pacificus researchers to analyze gene expression profiles in this organism with relative ease.

Methods
For smFISH staining, we followed the smFISH protocol for C. elegans described in WormBook (Ji and van Oudenaarden 2012).
For HCR, we followed the protocol described in Choi  a.For Mac: https://github.com/rwnull/HCRProbeMakerCL/tree/HCRProbeMakerCLforMacb.For PC: https://github.com/rwnull/insitu_probe_generator2. Probes were designed according to the guidelines described in Choi et al, 2016: the first probe in a pair began with an initiator sequence (B1, B2, or B3, see below), followed by the 2bp spacer 'tt', then a 25bp complimentary sequence.The second probe in a pair began with the adjacent complementary sequence, followed by the same spacer and initiator sequence.The following adaptations were made for the aforementioned experiments: a. Probe sets were designed such that the maximum number of probes were generated per gene.b.Initiator sequences were chosen such that Ppa-unc-25 could be uniquely labeled in a different color with every other gene investigated.

Gene Number of probe pairs Initiator sequence used (conjugated fluorophore)
Ppa Fixation: NOTE: Reagents, equipment & bench space were kept diligently RNAse free throughout.
1. Worms were washed off plates with 1 mL of M9 buffer and transferred to a 15 mL conical tube.To ensure high yield (worms will be lost throughout successive washes/incubations), at least 4 crowded plates of mixed developmental stages were used per sample.
2. Worms were centrifuged at 2000 x g for 2 min to bring worms to the bottom of the conical and M9 buffer was removed.
NOTE: For centrifugation speed, minimum possible speed needed to pellet worms was used.Density of liquid changes throughout protocol, so speed was optimized accordingly.
3. Worms were washed 3 times with 1 mL of M9 buffer each, then centrifuged at 2000 x g for 2 min between washes.
5. 1 mL of fresh paraformaldehyde (PFA) was added to each tube, on ice.Samples were immediately frozen at -80˚C overnight (>12h) before use.
NOTE: Worms can stay in -80˚C for long-term storage.Up to two weeks was attempted with minimal effect on quality of results.
6. Worms were fixed by thawing at room temperature for 45 minutes.This was done on a nutator for best results.
7. Worms were washed 2 times with 1 mL of PBST each.Worms were then centrifuged at 2000 x g for 2 min in between washes.
8. Worms were incubated in 1 mL of proteinase K (100 µg/mL) for 15 minutes at 37˚C on a nutator.Proteinase K concentration and treatment time was re-optimized for each batch of proteinase K and for samples at different developmental stages.Longer incubation period will allow for better probing of adult animals, for example.NOTE: Samples were imaged using a standard upright compound light microscope (equipped with the necessary filters) as soon as possible for best results.

Reagents
Probe Hybridization Buffer, Probe Wash Buffer, and Amplification Buffer were ordered from molecularinstruments.com.

Figure 1 .
Figure 1.smFISH and in situ HCR in P. pacificus: Cellular identities shown in all panels are tentative and are based on the identity of C. elegans neurons that express the respective gene in roughly the same position.Further confirmation is required to solidify these assignments.All images shown are maximum z-stack projections.Scale bar = 50µm.A: smFISH showing expression of Ppa-unc-25 in a J2 larval animal.AB/PB: location of anterior/posterior bulb of pharynx.B: HCR probing of Ppa-unc-25 (left) and Ppa-unc-30 (right) gene expression in a J2 larval animal.C,D: HCR probing of Ppa-unc-17 and Ppa-unc-25 gene expression in J2 (C) and J3 (D) larval animals.E,F: HCR probing of Ppa-eat-4 and Ppa-unc-25 gene expression patterns in J3 larval animals.

4 Fill
up to I L with ultrapure H 2 O Sterilize by autoclaving 4% Paraformaldehyde (PFA) For 40 mL of solution: 5 mL of 32% PFA solution 4 mL of 10x PBS Fill up to 40 mL with NF-free H 2 O PBST (1% Tween 20) For 50 mL of solution: 5 mL of 10x PBS 500 µL of Tween 20 Fill up to 50 mL with NF-free H 2 O Proteinase K solution (100µg/mL) For 1 mL of solution: 5 µL of 20 mg/mL proteinase K Fill up to 1 mL with PBST Store at -20˚C Glycine solution (2mg/mL) For 50 mL of solution: 100 mg of glycine Fill up to 50 mL with PBST Store at 4˚C 5x SSCT (1% Tween 20) For 40 mL of solution: 10 mL of 20x SSC 7/26/2024 -Open Access ; Shakes et al. 2012; Gendrel et al. 2016), which tend to distort and disintegrate the animal, particularly in adults, and therefore complicate the accurate visualization of gene expression patterns.